Method and kit for imaging and treating organs and tissues

ABSTRACT

Provided are methods and compositions for detecting and treating normal, hypoplastic, ectopic or remnant tissue, organ or cells in a mammal. The method comprises parenterally injecting a mammalian subject, at a locus and by a route providing access to said tissue or organ, with an composition comprising antibody/fragment which specifically binds to targeted organ, tissue or cell. The antibody/fragment may be administered alone, or labeled or conjugated with an imaging, therapeutic, cytoprotective or activating agent.

RELATIONSHIP TO OTHER APPLICATIONS

This application is a continuation of application Ser. No. 07/866,789,filed Apr. 7, 1992 now pending which is a continuation-in-part of Ser.No. 07/167,077 filed Mar. 11, 1988 now U.S. Pat. No. 5,101,327 which isa continuation of Ser. No. 06/1751,877 filed Jul. 5, 1985 now U.S. Pat.No. 4,735.210.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for imaging and treatinghypoplastic, absent, anatomically displaced or ectopic tissues andorgans and to a kit suitable for use therefor.

The invention also relates to methods for detecting the condition ofadjacent organs and detecting tissue retained after surgical removal ofan organ.

The invention also relates to methods of treating and ablatinghypoplastic and ectopic tissues or normal tissues.

The invention also relates to methods for protecting normal tissueduring radiation or chemotherapy of cancer, and composition and kitsuseful in the method.

2. Description of the Prior Art

There is a need for a method that clearly delineates hypoplastic,absent, anatomically displaced or ectopic tissue or organs and for amethod for treating conditions involving such tissues or organs.

It is important in certain clinical situations to detect the presence orabsence of particular tissues or organs. Moreover, it is often necessaryto determine whether an organ is anatomically correct and whether it haspathology by a non-invasive technique. It would be desirable to have anorgan imaging method using organ-specific imaging agents that would makeit possible to obtain a "positive" image of the organ, when normal, anda defect in organ visualization if pathology is present. Such a methodwould provide a new approach to scintigraphic and magnetic resonanceimaging of organs and tissues in the body based upon their immunologicalspecificity.

Normal tissues and organs have been imaged by magnetic resonance imagingtechniques, but not with the use of imaging-enhancing contrast agents,and not with antibody-conjugated imaging agents.

Methods of imaging tumors and infectious lesions using labeledantibodies and antibody fragments which specifically bind markersproduced by or associated with tumors or infectious lesions have beendisclosed, inter alia, in Hansen et al., U.S. Pat. No. 3,927,193 andGoldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457,4,444,744, 4,460,459 and 4,460,561, and in related pending applicationsU.S. Ser. Nos. 609, 607 and 633,999, the disclosures of all of which areincorporated herein in their entireties by reference. See also DeLand etal., J. Nucl. Med., 20, 1243-50(1979).

These methods use radiolabeled antibodies which specifically bind tomarkers produced by or associated with tumors or infectious lesions, andresult in a "positive" image, i.e., uptake of radioactivity attached tothe antibody in the structure involved with tumor or infectious lesionand having the appropriate antibody target, thus permitting avisualization of the involved structure. Further improvements in thespecificity and resolution of these methods is achieved by the use ofvarious substraction techniques which are also disclosed in theaforementioned references, and which enable background, non-specificradioactivity to be distinguished from specific uptake by the tumor orlesion.

Antibody conjugates comprising organ-specific and tissue-specificantibodies and addends for scintigraphic detection or magnetic resonanceimage enhancement have not been used as organ imaging reagents.

A need continues to exist for imaging and therapeutic methods which aremore sensitive and specific and for organ imaging and therapeuticreagents and methods with high specificity for differentiation ofparticular organs and tissues from surrounding-structures.

OBJECTS OF THE INVENTION

One object of the present invention is to provide a method for obtainingscintigraphic images that permits higher resolution and greaterspecificity for hypoplastic, absent, anatomically displaced or ectopictissues and organs.

Another object of the invention is to provide organ- and tissue-specificmethods and agents for scintigraphic and magnetic resonance imaging.

Another object of the invention is to provide reagents and kits suitablefor use in the imaging methods of the invention.

Another object of the invention is to provide methods for detecting thecondition of adjacent organs and detecting tissue retained aftersurgical removal of an organ.

Another object of the invention is to provide methods and agents fortreatment of normal organs and tissues that are hypoplastic, remnants,or anatomically displaced.

Another object of the invention is to provide methods and agents forablating normal cells and tissues as part of a therapeutic intervention.

Another object of the invention is to provide methods for protectingnormal tissue during radiation or chemotherapy of cancer, andcomposition and kits useful in the method.

Another object of the invention is to provide methods for altering thefunctions of a cell or tissue, especially hormone end organs.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an imaging method for positiveimaging of an hypoplastic, absent, anatomically displaced or ectopictissue or organ in a mammal. The method comprises the steps of (a)parenterally injecting a mammalian subject, at a locus and by a routeproviding access to said tissue or organ, with an amount of ascintigraphic imaging agent or magnetic resonance image enhancing agentsufficient to permit a scintigraphic image or an enhanced magneticresonance image of said structure to be effected; and (b) obtaining ascintigraphic image or an enhanced magnetic resonance image of saidstructure, at a time after injection of said agent sufficient for saidagent to accrete in said structure. The imaging agent comprises anantibody/fragment which specifically binds to said organ or tissue, andwhich is labeled with a radioisotope or a magnetic resonance enhancingagent.

In another embodiment, the invention provides a method for treatinghypoplastic, ectopic or remnant tissue or organ in a mammal. The methodcomprises parenterally injecting a mammalian subject, at a locus and bya route providing access to said tissue or organ, with apharmaceutically effective amount of an antibody/fragment whichspecifically binds to said organ or tissue. The antibody/fragment isconjugated with a therapeutic agent.

In another embodiment, the invention provides a method for detectingorgan tissue retained after surgical removal of a portion of the organ,wherein the organ tissue produces or is associated with a markersubstance. The method comprises injecting a human subject parenterallywith an antibody specific to the marker substance and radiolabeled witha pharmacologically inert radioisotope capable of detection using aphotoscanning device.

In another embodiment, the invention provides a method for determiningin a mammal, a first organ's condition and integrity, the first organproducing or being associated with a marker substance and being adjacentto a second organ which has been surgically removed from the mammal. Themethod comprises injecting the mammal parenterally with an antibodyspecific to the marker substance and radiolabeled with apharmacologically inert radioisotope capable of detection using aphotoscanning device.

In another embodiment, the invention provides a method for treatinghypoplastic or ectopic tissue which produces or is associated with amarker substance. The method comprises injecting a human subjectparenterally with an antibody specific to the marker substance andconjugated with a cytotoxic agent.

In another embodiment, the invention provides a method for destroyingbone-marrow cells in a patient prior to regrafting with normal bonemarrow cells. The method comprises treating the patient with a cytotoxicamount of an antibody or antibody fragment specific to a markerassociated with or produced by bone marrow cells and which is conjugatedto a cytotoxic agent.

In another embodiment, the invention provides an improved method oftherapy of cancer, wherein a human patient suffering from a cancersusceptible to treatment with radiation or a cytotoxic agent is treatedwith a therapeutic amount of radiation or a cytoxic agent. Theimprovement comprises administering to the patient acytotoxic-protective agent conjugated to an antibody or antibodyfragment which specifically binds to an antigen which is produced by orassociated with a normal cell.

In another embodiment, the invention provides a method of afftecing afunction of a non-malignant cell in a mammalian sugject, the methodcomprising administering to the subject a composition comparing anantibody specific to a growth factor receptor or hormone receptor on thetargeted cell, wherein the antibody affects the function andproliferation of the cell.

In another embodiment, the invention provides a method of treating acondition affecting non-malignant cells in a mammalian subject, themethod comprising administering to a subject requiring such treatment, acomposition comprising an antibody or fragment specific to a hormonereceptor or growth factor on a targeted cell, wherein the antibody orfragment is conjugated to a therapeutic agent.

In another embodiment, the invention provides an immunological method ofaffecting a hormonal function comprising administering to the subject acomposition comprising an antibody or fragment specific to a hormonereceptor on a targeted cell, wherein the antibody or fragment affectsthe hormonal function of the targeted cell.

In another embodiment, the invention provides an immunological method ofablating a cell in a mammalian subject, the method comprisingadministering to the subject requiring ablation of cell, a compositioncomprising an antibody or fragment specific to a hormone receptor orgrowth factor receptor on a cell targeted for ablation, wherein theantibody or fragment is conjugated to a chemical or radiation ablationagent.

Compositions and reagents and kits useful for practicing the methods ofthe invention are also provided.

DETAILED DESCRIPTION OF THE INVENTION

The above methods are beneficial for imaging and/or, where appropriate,treatment,

(1) hypoplastic or absent tissue or organs, in conditions such as,juvenile diabetes, wherein the islet cells of the pancreas can beatrophic or significantly reduced; thymic aplasia or agenesis;DiGeorge's Syndrome wherein there is a hypoplasia or absence ofparathyroid and the thymus,

(2) ectopic tissue and organs, such as, implants of endometrial glandsand stroma,

(3) retained tissue, such as, retained placental tissue after pregnancy,and organ remnants after surgical removal of the organ, and

(4) the condition of organs adjacent to a surgically removed organ.

(5) ablation of certain normal organs and tissues for other therapeuticpurposes, such as the spleen in patients with immune disease orlymphomas, the bone marrow in patients requiring bone marrowtransplantation, or normal cell types involved in pathologicalprocesses, such as certain T-lymphocytes in particular immune diseases.

The above methods include the use of a growth factor receptor antibodyor a hormone receptor antibody to target to end-organs bearing suchreceptor(s), the functions of which can be blocked with said antibodies.An isotopic or drug conjugate of these antibodies can also be used todeliver a therapeutic agent to said tissues and organs, in order toaffect diseases of tissues which bear such receptors. For example, inendometriosis, involving ectopic endometrial tissue, the currentstandard drug therapy involves administration of a synthetic steroidderived from ethisterone (DANOCRINE brand of danazol), which ischemically a 17-alpha-Pregna-2,3-dien-20-yno!2,3,3-d!-isoxazol-17-ol.This probably acts, at least in part, on sex steroid metabolism and withsex hormone receptors, particularly follicle-stimulating hormone (FSH)and luteinizing hormone (LH) at the target organ. It is now possible touse an antibody against these gonadal steroid receptors, alone or as animmunoconjugate with isotopes, drugs, toxins, hormone antagonists,cytokines, autocrines, etc., to inactivate and make the ectopicendometrium atrophic.

The above methods include providing an immunological method of affectingovarian and other hormone end-organ function, such as to induceamenorrhea or sterility. By use of an ovarian-targeting antibody or anantibody to an ovarian-related hormone receptor, such as FSH receptor,either as unconjugated antibodies or as antibodies conjugated with atherapeutic principle, a relatively convenient and safe method ofblocking ovarian function and inducing atrophy at the end-organ can beachieved.

Many hormone and growth factor receptors are known, and frequently showsufficient organ and tissue proclivity to allow these to serve astargets for antibodies which, when bound to said receptors, affect thefunction of the tissues and result in an immunological or, by the use ofconjugates with drugs, a chemical ablation, or a radiation ablation whenused as a conjugate with therapeutic isotopes.

Another application is in the treatment of fibrocystic breast disease.An antibody to FSH receptor or to estrogen receptor can be given aloneor as an immunoconjugate with a therapeutic principle to decrease thefibrocystic disease and to control its symptoms.

Still another indication is in benign prostatic hyperplasia or prostaticcancer, where the use of an antibody against an androgen receptor canalone, or as a conjugate with a therapeutic principle (hormone end-organantagonist, cytotoxic drug, toxin, or isotope), can decrease theprostatic tissue proliferation.

Where normal organs or tissues are developed abnormally or are displacedin the body, or are insufficiently removed during ablative surgery, thetissue/organ-associated antibodies may be used as tissue-targetedvehicles for delivering therapeutic agents to said tissues in order toinduce their involution or chemical and/or isotopic ablation. Theantibodies or their fragments (or subfragments) can be conjugated withtherapeutic modalities including, but not limited to, isotopes, drugs,toxins, photodynamic therapy agents, cytokines, hormones, autocrines,etc., which are used as cytotoxic or modulating agents, and which havehitherto been employed principally as toxic conjugates tocancer-targeting antibodies, as described in the reviews by Waldmann, T.A., Science 252:1657, 1991; Koppel, G. A., Bioconjug. Chem. 1:13, 1990;Oeltmann, T. N., and Frankel, A. E., FASEB J. 5:2334, 1991; and van denBergh, H. E., chemistry in Britain, May 1986, 430-439, incorporatedherein in their entirety by reference.

Another therapeutic application for such organ- and tissue-targetingantibodies conjugated with a toxic agent is for the ablation of certainnormal cells and tissues as part of another therapeutic strategy, suchas in bone marrow ablation with antibodies against bone marrow cells ofparticular stages of development and differentiation, and in thecytotoxic ablation of the spleen in patients with lymphoma or certainimmune diseases, such as immune thrombocytopenic purpura, etc.

Another therapeutic application for such organ- and tissue-targetingantibodies or fragments is to conjugate them with a cytoprotectiveagent. The conjugate is administered to a patient undergoingchemotherapy or radiation therapy so that the targeted normal organs andtissues are protected during the therapy.

Several methods are known to those skilled in the art for producingorgan or tissue associated or specific antibodies, if existingantibodies are considered unsuitable or if different or morediscriminating specificities are desired. Generally, whole cells, tissuesamples and/or cell or tissue fractions, membranes, antigen extracts orpurified antigens are used to challenge the immune system of a suitableanimal, e.g., a mouse, rabbit, hamster, goat or the like, the antigenbeing rendered immunogenic by aggregation if necessary and/or bycoadministration with a suitable conventional adjuvant. Hyperimmuneantiserum can be isolated and polyclonal antibodies prepared byconventional procedures. Alternatively, spleen cells can be fused withimmortal myeloma cells to form hybridoma cells producing monoclonalantibodies, by what are now conventional procedures. See, e.g., theprocedures in the above-referenced U.S. Pat. application Ser. No.609,607 for illustrative techniques. Hybridomas using animal, e.g.,mouse, or human myeloma cell lines and animal or human spleen or lymphcells are all know in the art, and can be made and used for the presentmethod. See, for example, Glassy et al., "Human Monoclonal Antibodies toHuman Cancers", in "Monoclonal Antibodies and Cancer", Boss et al.,Eds., 163-170 (Academic Press, 1983). The specific antisera ormonoclonals are screened for specificity by methods used to screen theanti-lymphocyte clones in the references cited hereinabove, whichmethods are also conventional by now in this art.

Organ-associated and organ-specific antibodies can be developed byimmunizing a suitable animal host with certain mammalian tumors ornormal-organ/tissue extracts and/or cells, as well as with purifiedhormone receptors or growth factor receptors. It is well known that useof tumors as immunogens can result in antibodies which not only reactwith neoplasia but also with normal tissue components which sometimesshow an organ-restricted nature. Histogenetic and functional differencesbetween various tissues and organs of the body of course suggest thatdistinct antigens are present and identifiable. A body of scientificliterature already exists which claims the identification oforgan-specific antigens, either using classical immunization approachesor by immunizing with specific tumors, and this is reviewed byGoldenberg et al., Cancer Res., 3455(1976), showing that such antigensare known and available.

Similar organ- and tissue-associated and specific antigens areidentifiable by hybridoma methods which produce monoclonal antibodies.One recent development is the production of human hybridoma monoclonalantibodies by securing lymphocytes or plasma cells from patients showingcertain organ-restricted autoimmune diseases, e.g., thyroiditis,gastritis, ulcerative colitis, myositis, and the like. Theseantibody-producing cells are then fused in vitro with human or murinemyeloma cells and hybridomas of appropriate anti-organ and anti-tissueantibody formation are produced and propagated, using well knownmethods. Also, patients with specific tumor types can be used as asource of such lymphocytes or plasma cells, or such patients can befurther immunized with such tumor cells for stimulating the productionof anti-organ and anti-tissue antibodies. The lymphatic tissue removedis then used for fusion with suitable myeloma cells, by procedures whichare by now well known and conventional in the art.

Organ-associated and organ-specific antigens can be isolated forimmunization of another species, e.g., subhuman primates, rodents,rabbits, goats, etc., by a number of methods known in the art, such asisolation of cell membranes or disruption of the cells, e.g., bycentrifugation, sonication, etc., to obtain intracellular antigens. Itis preferable, for these purposes, to use intracellular as opposed tosurface and extracellular antigens. In this manner, organ-associated andorgan-specific antigens can be obtained from a large number of tissuesand organs of the body, including brain, thyroid, parathyroid, larynx,salivary glands, esophagus, bronchus and lungs, heart, liver, pancreas,stomach and intestines, kidney, adrenal gland, ovary, testis, uterus,prostate, etc. Of further interest is the differentiation of differenttissue and cellular components within an organ, such as tubular andglomerular kidney, different regions and cell types of the brain,endocrine and exocrine pancreas, etc., especially by the identificationof antigens and antigen epitopes restricted to the individual cell andtissue types in question, as accomplished with polyclonal and/orhybridoma-monoclonal antibody-production methods known in the art.

Antibodies can be produced using cells isolated from tissue obtained atautopsy. For example, mice can be immunized with such tissues for aperiod necessary to evoke anti-specific organ or tissue antibodies. Thespleens of these mice are removed and then fused, by standard methods,with a murine myeloma cell line suitable for hybridoma production. Usingmethods already standard in the art, monoclonal antibody-producinghybridomas are selected and propagated, and those with organ- ortissue-associated antibody production are cloned and expanded as asource of organ or tissue antibodies. Absolute tissue specificity is notrequired since significant quantitative differences ordinarily sufficefor operational specificity for imaging purposes.

Antibodies and fragments useful in the methods of the present inventioninclude those against antigens associated or produced by normal organs,tissues, and cells, and may or may not be cross-reactive with certainneoplastic tissues.

Preferred are those which, prior to being labeled or conjugated, have aspecific immunoreactivity to targeted cells, tissue or organs of atleast 60 % and a cross-reactivity to other antigens of less than 35 %.

Specific examples include antibodies and fragments against bone marrowcells, particularly hematopoietic progenitor cells, pancreatic isletcells, spleen cells, parathyroid cells, uterine endometrium, ovarycells, testicular cells, thymus cells, B-cells, T-cells, Null cells,vascular endothelial cells, bile duct cells, gall bladder cells,prostate cells, hormone receptors such as of FSH, LH, TSH, growth factorreceptors, such as of epidermal growth factor, urinary bladder cells,and vas deferens cells.

An example of a monoclonal antibody that reacts with granulocytes andwhich targets to bone marrow cells is NP- 2, disclosed in Sharkey etal., Cancer Res. 50:2823-283 (1990). This antibody is useful, whenconjugated with a cytotoxic agent, for ablating the bone marrow. Anothergranulocyte antibody, MN3, has similar properties. Other antibodies thatreact with other bone marrow cells, especially progenitor cell types,commercially available, are likewise suitable for bone marrow ablation.

Antibodies that target the spleen well include the LL2 (also known asEPB-2) monoclonal antibody, disclosed in Pawlak-Byczkowska, CancerResearch, 49:4568-4577 (1989), which is directed against normal andmalignant B-cells, and which can be used for treating normal spleencells in patients with immune diseases, lymphoma, and other diseases.

Antibodies specific to uterine endometrium are preferred for targetingand therapy of endometriosis, when conjugated with suitable diagnosticor therapeutic agents, respectively.

Antibodies useful in the present invention may be whole immunoglobulinof any class, e.g., IgG, IgM, IgA, IgD, IgE, chimeric or hybridantibodies with dual or multiple antigen or epitope specificities. Itcan be a polyclonal antibody, preferably an affinity-purified antibodyfrom a human or an appropriate animal, e.g., a primate, goat, rabbit,mouse or the like. Monoclonal antibodies are also suitable for use inthe present method, and are preferred because of their highspecificities. They are readily prepared by what are now consideredconventional procedures of immunization of mammals with immunogenicantigen preparation, fusion of immune lymph or spleen cells with animmortal myeloma cell line, and isolation of specific hybridoma clones.More unconventional methods of preparing monoclonals antibodies are notexcluded, such as interspecies fusions and genetic engineeringmanipulations of hypervariable regions, since it is primarily theantigen specificity of the antibodies that affects their utility in thepresent invention. It will be appreciated that newer techniques forproduction of monoclonals can also be used, e.g., human monoclonals,interspecies monoclonals, chimeric (e.g., human/mouse) monoclonals,genetically engineered antibodies and the like.

Antibody fragments useful in the present invention are F(ab')₂, F(ab)₂,Fab', Fab, Fv and the like, including hybrid fragments. Also useful areany subfragments retaining the hypervariable, antigen-binding region ofan immunoglobulin and having a size smaller than a Fab' fragment. Thiswill include genetically engineered and/or recombinant proteins,whether, single-chain or multi-plechain, which incorporate an antigenbinding site and otherwise function in vivo as targeting vehicles insubstantially the same way as natural immunoglobulin fragments. Suchsingle-chain binding molecules are disclosed in U.S. Pat. No. 4,946,778,which is hereby incorporated by reference. Fab' antibody fragments maybe conveniently made by reductive cleavage of F(ab')₂ fragments, whichthemselves may be made by pepsin digestion of intact immunoglobulin. Fabantibody fragments may be made by papain digestion of intactimmunoglobin, under reducing conditions,, or by cleavage of F(ab)₂fragments which result from careful papain digestion of whole Ig. Thefragments may also be produced by genetic engineering.

It should be noted that mixtures of antibodies, isotopes, andimmunoglobulin classes can be used, as can hybrid antibodies. Thehybrids can have two different antigen specificities, e.g., one armbinding to one organ antigen and another arm binding to another antigen,or one arm could bind to one epitope on the antigen, and the other armcould bind to another epitope. The foregoing are merely illustrative,and other combinations of specificities can be envisioned that also fallwithin the scope of the invention.

Hybrid antibody fragments with dual specificities can be preparedanalogously to the anti-tumor marker hybrids disclosed in U.S. Pat. No.4,361,544. Other techniques for preparing hybrid antibodies aredisclosed in, e.g., U.S. Pat. No. 4,474,893, U.S. Pat. No. 4,479,895,U.S. Pat. No. 4,714,681, U.S. Pat. No: 4,474,893 and in Milstein et al.,Immunol. Today, 5,299(1984), all incorporated herein by reference.

The methods of the present invention include the use of pretargetedantibody methods, and the use of light with porphyrins and fluorescentdyes. The methods taught in the prior art are utilized in cancertherapy. However, if the antibody fragment utilized is targeted toorgans and tissues specified herein, analogous procedures may be used inthe present invention.

For example, Paganelli, Nucl. Med. Commun. 12:211, 1991, incorporatedherein in its entirety by reference, disclosed antibody pretargetingprocedures, such as using streptavidin-conjugated antibodies,biotinylated antibodies in conjunction with avidin and biotin,bifunctional antibodies, antibody-hapten complexes, andenzyme-conjugated antibodies, in addition to delivering radiation totarget cells and tissues by such 2- and 3-step procedures.

When the cell or tissue is pretargeted by a 2- or 3-step procedure, thesubject is injected with a first composition comprising, for example, astreptavidin-conjugated antibody, biotinylated antibody to be used inconjunction with avidin and biotin, bifunctional antibody,antibody-hapten complexes, or enzyme-conjugated antibody, wherein theantibody is an antibody or antibody fragment which specifically binds amarker produced by or associated with said cell or tissue. After thefirst composition accretes at the targeted tissue or cell, a secondcomposition, which bears the desired imaging, therapeutic,cytoprotective or activating principle, is administered. The secondcomposition either activates the first composition or couples with thefirst composition to produce a desired effect.

When the cell or tissue is pretargeted in a 3-step procedure, thesubject is injected with the first composition which comprisesbiotinylated antibody or fragment, is then injected with a clearingcomposition comprising an agent to clear circulating biotinylatedantibody or fragment, and then injected with the second compositionwhich comprises biotin conjugated with the desired imaging, therapeutic,cytoprotective or activating agent.

When the term "antibody" is used herein, all the above types ofantibodies and fragments are included therein.

The use of light and porphyrins in cancer therapy has been reviewed byvan den Bergh (Chemistry in Britain, May 1986, Vol. 22, pp. 430-437),which is incorporated herein in its entirety reference, and includesreference to the use of monoclonal antibodies conjugated with aphotosensitizer for transporting the latter to the tumor. This has beensuggested earlier by Oseroff in Photochem. Photobiol. 41:35S, 1985; Mewet al., Cancer Res. 45:4380, 1985; Hasan et al., Immunity to Cancer, II,pp. 471-477, 1989 Alan R. Liss, Inc. publishers!; and Pelegrin et al.,Cancer 67:2529, 1991 all incorporated herein by reference!, whichinvolved tissue culture or animal studies of fluorescent dyes attachedto antitumor antibodies.

Radiolabeled antibodies to markers characteristic of hypoplastic,absent, displaced or ectopic tissues or organs are a new kind of agent.They are an example of an immunologic, organ-specific imaging agentwhich can be used to ascertain the location and shape of a specificorgan and reveal possible abnormalities therein. Such agents are usefulfor imaging organs such as, e.g., liver, spleen, pancreas, and the like,and many antibodies which specifically bind to tissues of these organsare known and/or under current investigation and development.

Among the radioisotopes used, gamma-emitters, positron-emitters, x-rayemitters and fluorescence-emitters are suitable for localization and/ortherapy, while beta-emitters and alpha-emitters may also be used fortherapy. Suitable radioisotopes for the methods of the present inventioninclude: Astatine-211, Iodine-123, Iodine-125, Iodine-126, Iodine-131,Iodine-133, Bismuth-212, Bromine-77, Indium-111, Indium-113m,Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium-103,Ruthenium-105, Mercury-107, Mercury-203, Rhenium-186, Rhenium-188,Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165,Thulium-167, Thulium-168, Technetium-99m, Fluorine-18, Silver-111,Platinum-197, Palladium-109, Copper-67, Phosphorus-32, Phosphorus-33,Yttrium-90, Scandium-47, Samarium-153, Lutetium-177, Rhodium-105,Praseodymium-142, Praseodymium-143, Terbium-161, Holmium-166, Gold-199,Cobalt-57, Cobalt-58, Chromium-51, Iron-59, Selenium-75, Thallium-201,and Ytterbium-169. Preferably the radioisotope will emit in the 10-5,000kev range, more preferably 50-1,500 kev, most preferably 50-500 kev.

Isotopes preferred for diagnostic use include Iodine-123, Iodine-131,Indium-111, Gallium-67, Ruthenium-97, Technetium-99m, Copper-67,Cobalt-57, Cobalt-58, Chromium-51, Iron-59, Selenium-75, Thallium-201,and Ytterbium-169.

Isotopes preferred for therapeutic use include: Iodine-125, Iodine-131,Rhenium-186, Rhenium-188, Silver-111, Platinum-197, Palladium-109,Copper-67, Phosphorus-32, Phosphorus-33, Yttrium-90, Scandium-47,Samarium-153, Lutetium-177, Rhodium-105, Praseodymium-142,Praseodymium-143, Terbium-161, Holmium-166, and Gold-199.

Many drugs and toxins are known which have cytotoxic effects on cells.They are to be found in compendia of drugs and toxins, such as the MerckIndex, Goodman and Gilman, and the like, and in the references citedabove. Any such drug can be conjugated to or loaded onto the antibody byconventional means well know in the art, and illustrated by analogy tothose described above.

The present invention also contemplates dyes used, for example, inphotodynamic therapy, conjugated to the antibodies and fragments, andused in conjunction with appropriate nonionizing radiation.

Examples of known cytotoxic agents useful in the present invention arelisted in Goodman et al., "The Pharmacological Basis of Therapeutics,"Sixth Edition, A. G. Gilman et al, eds./ Macmillan Publishing Co. N.Y.,1980. These include taxol, nitrogen mustards, such as, mechlorethamine,cyclophosphamide, melphalan, uracil mustard and chlorambucil;ethylenimine derivatives, such as, thiotepa; alkyl sulfonates, such as,busulfan; nitrosoureas, such as, carmustine, lomustine, semustine andstreptozocin; triazenes, such as, dacarbazine; folic acid analogs, suchas, methotrexate; pyrimidine analogs, such as, fluorouracil, cytarabineand azaribine; purine analogs, such as, mercaptopurine and thioguanine;vinca alkaloids, such as, vinblastine and vincristine; antibiotics, suchas, dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin andmitomycin; enzymes, such as, L-asparaginase; platinum coordinationcomplexes, such as, cisplatin; substituted urea, such as hydroxyurea;methyl hydrazine derivatives, such as, procarbazine; adrenocorticalsuppressants, such as, mitotane; hormones and antagonists, such as,adrenocortisteroids (prednisone), progestins (hydroxyprogesteronecaproate, medroprogesterone acetate and megestrol acetate), estrogens(diethylstilbestrol and ethinyl estradiol), antiestrogens (tamoxifen),and androgens (testosterone propionate and fluoxymesterone).

The antibodies and fragments of the invention may be radiolabeled by avariety of methods known in the art. Many of these methods are disclosedin the above-referenced U.S. Patents and Patent Applications, andinclude direct radioiodination. See also, Rayudu, op, cit.; and Childset al., J. Nuc. Med., 26, 293(1985). Any conventional method ofradiolabeling which is suitable for labeling isotopes for in vivo usewill be generally suitable for labeling imaging agents according to thepresent invention.

The antibodies and fragments may be conjugated to therapeutic agentssuch as drugs, toxins, boron addends, isotopes, fluorescent dyesactivated by nonionizing radiation, hormones, autocrines, cytokines,cytoprotective agents, etc., by methods known to those skilled in theart. U.S. Pat. No. 5,057,313, Shih et al, hereby incorporated byreference, teaches one such method.

Loading of drugs on to a carrier as disclosed in U.S. Pat. No. 5,057,313will depend upon the potency of the drug, the efficiency of the antibodytargeting and the efficacy of the conjugate once it reaches its target.In most cases., it is desirable to load at least 20, preferably 50 andoften 100 or more molecules of a drug on a carrier. The ability topartially or completely detoxify a drug as a conjugate, while it iscirculation, can reduce systemic side effects of the drug and permit itsuse when systemic administration of the unconjugated drug would beunacceptable.

Toxins will often be less heavily loaded than drugs, but it will stillbe advantageous to load at least 1, preferably 5, and in some cases 10or more molecules of toxin on a carrier and load at least one carrierchain on the antibody for targeted delivery.

The conjugate will generally be administered as a sterile aqueoussolution in phosphate-buffered saline. Dosage will depend on thetherapeutic utilized, the desired effect and the side effectsexperienced by the patient.

The imaging agent will normally be administered at a site and by a meansthat insure that it is mobilized and taken up into the organ or tissuewhich will vary by the tissue or organ to be imaged.

The agent is preferable injected by a systemic route, e.g.,intravenously, intraarterially, intramuscularly or subcutaneously, or bya combination of systemic routes insuring its accretion in the tissue ororgan of interest.

Volumes of labeled antibody imaging agent, normally in sterilephysiological saline, will normally vary somewhat depending upon thesite, the concentration, and the number of injections. Activity of theagent will normally be in the range of about 10-40, preferably about15-25 mCi per injection for a Tc-99m-labeled agent. It will beappreciated that the activity will vary for other radioisotopes,depending upon their half-lives, their imaging characteristics, i.e.,energy ranges, emission intensities, scatter and the like, the stabilityof the labeled agent, especially antibody conjugates, their distributionand clearance, and the time at which imaging is to be done. Adjustmentof these parameters will be conventional for the ordinary skilledclinician.

Imaging is normally effected up to about 24 hours, more preferably atabout 2-6 or less hours after injection of the imaging agent, to obtainthe "positive" image of the organ or tissue. Two- and three-steptargeting methods will require longer periods.

Timing of injections of imaging agents will depend upon the types ofagents and methods used and the targeting patterns to the organs andtissues of interest.

The conjugate will generally be administered as a sterile aqueoussolution in a buffered saline. Dosage units of about 1-200 mg ofconjugate will be administered for a duration of treatment as determinedby the skilled practitioner. It may be necessary to reduce the dosageand/or use antibodies from other species and/or hypoallergenicantibodies, e.g., chimeric mouse/human, CDR-grafted ("humanized"), orprimate antibodies, to reduce patient sensitivity.

Routes of administration include intravenous, intraarterial,intrapleural, intraperitoneal, intrathecal, subcutaneous or byperfusion.

An application of the organ- or tissue-specific or organ-ortissue-associated antibodies disclosed hereinabove is for normal organscintigraphy and magnetic resonance imaging (mri). In this case, asuitably radiolabeled antibody/fragment or an antibody/fragment bearinga mr image enhancing agent is administered with the intention ofobtaining a "positive" image of the organ, when normal, and a defect inorgan visualization if pathology is present. This provides a newapproach to organ- and tissue-specific nuclear and magnetic resonanceimaging of organs and tissues in the body, based upon theirimmunological specificity.

The method of the invention can be practiced either with scintigraphicor magnetic resonance imaging agents. A combination of these imagingagents can also be used, although this requires more complexinstrumentation and data processing.

Scintigraphic imaging according to the method of the invention iseffected by obtaining a scintigram of the tissue or organ of interest,using as an imaging agent a radiolabeled antibody which specificallybinds to a marker produced by or associated with the tissue or organ orat a locus proximal to the tissue or organ and draining into thestructure, such that the antigen/marker accretes in discrete focitherein.

The scintigram is normally taken by a gamma imaging camera having one ormore windows for detection of energies in the 50-500 keV range. Use ofradioisotopes with higher energy, beta, or positron emissions wouldentail use of imaging cameras with the appropriate detectors, all ofwhich are conventional in the art.

The scintigraphic data can be stored in a computer for later processing.

Magnetic resonance imaging (mri) is effected in an analogous manner toscintigraphic imaging except that the imaging agents will containmagnetic resonance (mr) enhancing species rather than radioisotopes. Itwill be appreciated that the magnetic resonance phenomenon operates on adifferent principle from scintigraphy. Normally, the signal generated iscorrelated with the relaxation times of the magnetic moments of protonsin the nuclei of the hydrogen atoms of water molecules in the region tobe imaged. The magnetic resonance image enhancing agent acts byincreasing the rate of relaxation, thereby increasing the contrastbetween water molecules in the region where the imaging agent accretesand water molecules elsewhere in the body. However, the effect of theagent is to decrease both T₁, and T₂, the former resulting in greatercontrast while the latter results in lesser contrast. Accordingly, thephenomenon is concentration-dependent, and there is normally an optimumconcentration of a paramagnetic species for maximum efficacy. Thisoptimal concentration will vary with the particular agent used, thelocus of imaging, the mode of imaging, i.e., spin-echo,saturation-recovery, inversion-recovery and/or various other strongly T₁-dependent or T₂ -dependent imaging techniques, and the composition ofthe medium in which the agent is dissolved or suspended. These factors,and their relative importance are known in the art. See, e.g., Pykett,Scientific American, 246, 78(1982); Runge et al., Am. J. Radiol., 141,1209(1983).

The mr image enhancing agent must be present in sufficient amounts toenable detection by an external camera, using magnetic field strengthswhich are reasonably attainable and compatible with patient safety andinstrumental design. The requirements for such agents are well known inthe art for those agents which have their effect upon water molecules inthe medium, and are disclosed, inter alia, in Pykett, op. cit., andRunge et al., op. cit.

Preparation of antibodies conjugated to a magnetic resonance imageenhancing agent can be effected by a variety of methods. In order toload an antibody molecule with a large number of paramagnetic ions, itmay be necessary to react it with a reagent having a long tail to whichare attached a multiplicity of chelating groups for binding the ions.Such a tail can be a polymer such as a polylysine, polysaccharide, orother derivatized or derivatizable chain having pendant groups to whichcan be bound chelating groups such as, e.g., ethylenediaminetetraaceticacid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and thelike groups known to be useful for this purpose. The chelate is normallylinked to the antibody by a group which enables formation of a bond tothe antibody with minimal loss of immunoreactivity and minimalaggregation and/or internal cross-linking. Other, more unusual, methodsand reagents for conjugating chelates to antibodies are disclosed incopending U.S. Patent application Ser. No. 742,436 to Hawthorne,entitled "Antibody Conjugates", filed Jun. 7, 1985, the disclosure ofwhich is incorporated herein in its entirety by reference.

MRI contrast agents are well known in the art and include, for example,Gadolinium, Iron, Manganese, Rhenium,, Europium, Lanthanium, Holmium,and Ferbium.

The mr scans are stored in a computer and the images processedanalogously to the scintigraphic data.

Several agents are known to those skilled in the art which protectnormal cells, tissues and organs during treatment of cancer withradiation and cytotoxic agents. Such agents are disclosed in "CancerChemotherapy--Principles & Practice", Chabner et al. eds., J. B.Lippincott Company, Philadelphia, 1990, especially Chapter 18,"Radiation and Chemotherapy Sensitizers and Protectors" by Coleman etal., which is incorporated herein by reference.

These agents include WR-2721 (S2-3-aminopropylamino!ethylphosphorothioic acid), known as Ethyol beingdeveloped by US Bioscience, WR-2721's dephosphorylated metabolite,WR-1065, and MGI 136 being developed by MGI Pharma Inc.

The reagents are conveniently provided in kit form, adapted for use inthe methods of the invention. Kits will normally contain separate sealedsterile vials of injectable solutions of labeled reagents, orlyophilized antibodies/fragments or antibody/fragment conjugates andvials of sterile conventional injection vehicles with which they will bemixed just prior to administration.

Kits may also include reagents for labeling antibodies, e.g.,Chloramine-T (for I-131 or I-123 labeling), SnCl₂ (for Tc-99m labelingusing pertechnetate from a commercial generator), short columns forsizing and/or purification of reagents, and other conventional accessorymaterials.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. In the followingexamples, all temperatures are set forth uncorrected in Celsius; unlessotherwise indicated, all parts and percentages are by weight.

EXAMPLES Example 1--Imaging Pancreatic Cells

Hybridoma-monoclonal antibodies are made in the mouse to the Langerhanscells of the endocrine pancreas, derived from a human autopsy specimumshortly after death. The monoclonal F(ab')₂ reactive against the antigenepitope showing relatively high specificity for Langerhans cells of thepancreas, as demonstrated, e.g., by immunohistology, are labeled with agamma-emitting isotope, such as with I-123, and injected, e.g. 0.15 mgmonoclonal against endocrine pancreas antigen, labeled usingChloramine-T with I-123, at a dose of 3.0 mCi, injected i.v. in a3-month-old male suspected of having pathology of the endocrinepancreas. External gamma-camera imaging is performed at 6, 24, and 48hours after injection, without substraction. In this specific case,decreased to almost absent accretion of I-123 radioactivity in thepancreas is suggestive of endocrine pancreas pathology in an infantpresenting with pancreas hormone deficiency shortly after birth.

Example 2--Bone Marrow Ablation and Cancer Therapy

A middle-age woman with advanced breast cancer, including bone and bonemarrow invasion, has an aliquot of her bone marrow removed and harvestedfor regrafting after clearing the marrow of the cancer cells in vitro.The bone marrow in the patient is then destroyed by i.v. infusion of 20mg NP-2 monoclonal antibody F(ab')₂ labeled with 200 mCi Rhenium-188according to the method of Griffiths et al. (Cancer Res. 51:4594, 1991).Approximately 3 weeks later, there is evidence of severe bone marrowtoxicity which requires the infusion of the autologous bone marrow whichwas previously cleared of cancer cells,- in combination withhematopoietic growth factor administration, in this case with GM-CSFgiven repeatedly before and after marrow grafting. Six weeks later, thepatient has renewed bone marrow function and an MN3-Fab' (Tc-99m) bonemarrow scan shows good bone marrow imaging without evidence ofmetastatic defects. She is now a candidate for treatment of other sitesof her metastatic breast cancer.

Example 3--Endometriosis Detection

A woman complains of amenorrhea and infertility and is suspected ofhaving endometriosis. She is injected with 1 mg of anti-endometrialtissue monoclonal antibody Fab' labeled with Tc-99m (20 mCi)intravenously. Four hours later, a total body planar scan revealsabnormal foci of radioactivity in the right lower chest and in theretroperitoneum, which are confirmed by single photon emission computertomography immediately thereafter. The patient is then referred toablation therapy.

Example 4--Endometriosis Therapy

A woman is diagnosed to have endometriosis and is referred to hergynecologist for treatment. An endometrial tissue-associated monoclonalantibody IgG and a monoclonal antibody IgG against FSH receptor arelabeled with I-131 by the chloramine-T method at a specific activity of10 mCi/mg, and the combination is then infused i.v. to deliver a dose of100 mCi I-131. After monitoring her peripheral blood cells during thenext month, a repeat therapy is given 6 weeks later. After an additional6 weeks, the patient shows a complete remission of her symptoms.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

It will be understood that the invention is not limited to use of knownantibodies or markers, but can be practiced with antibodies to anymarker produced by or associated with an organ or tissue.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

I claim:
 1. A method of imaging anatomically displaced organs or ectopiccells or tissues in a mammalian subject by scintigraphic or magneticresonance imaging, comprising (a) parenterally injecting a mammaliansubject, at a locus and by a route providing access to an organ or tocells or tissues characteristic of said organ of interest, with anantibody or antibody fragment which specifically binds a marker producedby or associated with said cells or tissues, said antibody or antibodyfragment being labeled with a radioisotope or with a magnetic resonanceimage enhancing agent capable of external detection, the amount of thelabeled antibody or antibody fragment being sufficient to permit ascintigraphic or an enhanced magnetic resonance image of said organ,cells or tissue to be obtained; and (b) obtaining a positivescintigraphic image or positive enhanced magnetic resonance image ofsaid organ, cells or tissues, at a time after injection of said agentsufficient for said agent to specifically bind to said marker and, whereit is desired to observe the anatomical position of an organ, todiffusely accrete in said organ.
 2. The method of claim 1, wherein thecell or tissue is pretargeted with a first composition comprising astreptavidin-conjugated antibody, biotinylated antibody to be used inconjunction with avidin and biotin, bifunctional antibody,antibody-hapten complexes, or enzyme-conjugated antibody, wherein theantibody is an antibody or antibody fragment which specifically binds amarker produced by or associated with said cell or tissue, and after thefirst composition accretes at the targeted tissue or cell, a secondcomposition, which bears the imaging principle, is administered whichactivates the first composition or couples with the first composition toproduce a desired effect.
 3. The method of claim 2, wherein the cell ortissue is pretargeted by injecting the subject with a first compositionwhich comprises a biotinylated antibody or fragment or anavidin-conjugated antibody or fragment, optionally injecting the patientwith a clearing composition comprising an agent to clear circulatingbiotinylated or avidin-conjugated antibody or fragment, injecting avidinwhen the first composition is a biotinylated antibody, and theninjecting the second composition which comprises biotin conjugated withisotope or mri enhancing agent.
 4. The method of claim 1, wherein theisotope emits at 50-1,500 kev energy.
 5. The method of claim 1, whereinthe mri enhancing agent is a species of Gadolinium, Iron, Manganese,Rhenium, Europium, Lanthanium, Holmium or Ferbium.
 6. The method ofclaim 1, wherein the antibody is a Fv, single chain antibody, Fab, Fab',or F(ab')₂ fragment or intact antibody.
 7. The method of claim 1,wherein the antibody or fragment has a specific immunoreactivity totargeted cells or tissues of at least 60% and a cross-reactivity toother antigens of less than 35%.
 8. The method of claim 1, wherein saidcells or tissues to be imaged are anatomically displaced cells ortissues.
 9. The method of claim 1, wherein said cells or tissues to beimaged are ectopic cells or tissues.
 10. The method of claim 9, whereinsaid cells or tissues to be imaged are endometrial tissues.
 11. Themethod of claim 1, wherein the mri enhancing agent is a species ofGadolinium, Iron, Manganese, Rhenium, Europium, Lanthanum, Holmium orTerbium.
 12. The method of claim 1, wherein the antibody is a Fv, singlechain antibody, Fab, Fab', or F(ab')₂ fragment or intact antibody. 13.The method of claim 1, wherein the antibody or fragment has a specificimmunoreactivity to targeted cells or tissues of at least 60 % and across-reactivity to other antigens of less than 35 %.
 14. The method ofclaim 1, wherein said antibody or fragment is a monoclonal antibody orfragment.
 15. A method of imaging a hypoplastic organ or hypoplasticcells or tissues in a mammalian subject by scintigraphic or magneticresonance imaging, comprising (a) parenterally injecting a mammaliansubject, at a locus and by a route providing access to an organ or tocells or tissues of interest, with an antibody or antibody fragmentwhich specifically binds a marker produced by or associated with saidcells or tissues, said antibody or antibody fragment being labeled witha radioisotope or with a magnetic resonance image enhancing agentcapable of external detection, the amount of the labeled antibody orantibody fragment being sufficient to permit a scintigraphic or anenhanced magnetic resonance image of said organ, cells or tissues beobtained; and (b) obtaining a positive scintigraphic image or positiveenhanced magnetic resonance image of said organ, cells or tissues, at atime after injection of said agent sufficient for said agent tospecifically bind to said marker and, where it is desired to observehypoplasia of an organ, to diffusely accrete in said organ.
 16. Themethod of claim 15, wherein said imaging is used in diagnosis ofjuvenile diabetes.
 17. The method of claim 15, wherein said imaging isused in diagnosis of thymic aplasia or agenesis.
 18. The method of claim15, wherein said imaging is used in diagnosis of DiGeorge's syndrome.19. The method of claim 15, wherein the cell or tissue is pretargetedwith a first composition comprising a streptavidin-conjugated antibody,biotinylated antibody to be used in conjunction with avidin and biotin,bifunctional antibody, antibody-hapten complex, or enzyme-conjugatedantibody, wherein the antibody is an antibody or antibody fragment whichspecifically binds a marker produced by or associated with said organ,cells or tissues, and after the first composition accretes at thetargeted tissue or cell, a second composition, which bears the imagingprinciple, is administered which activates the first composition orcouples with the first composition to produce a desired effect.
 20. Themethod of claim 19, wherein the cell or tissue is pretargeted byinjecting the subject with a first composition which comprises abiotinylated antibody or fragment or an avidin-conjugated antibody orfragment, optionally injecting the patient with a clearing compositioncomprising an agent to clear circulating biotinylated oravidin-conjugated antibody or fragment, injecting avidin when the firstcomposition is a biotinylated antibody, and then injecting the secondcomposition which comprises biotin conjugated with isotope or mrienhancing agent.
 21. The method of claim 15, wherein the isotope emitsat 50-1,500 kev energy.
 22. The method of claim 15, wherein saidantibody or fragment is a monoclonal antibody or fragment.